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The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3'-5'-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activates specific pathways and mediates phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogues, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position more than a carbonyl at the C6 position. We also identified phosphate-modified analogues that bind both the ydaO RNA and GdpP protein with high affinity, whereas symmetrically modified ribose analogues exhibited a substantial decrease in ydaO affinity but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogues could be useful as chemical tools to specifically target subsections of second-messenger signaling pathways. [ABSTRACT FROM AUTHOR]

Knowledge of RNA structure is necessary to determine structure-function relationships and to facilitate design of potential therapeutics. RNA secondary structure prediction can be improved by applying constraints from nuclear magnetic resonance (NMR) experiments to a dynamic programming algorithm. Imino proton walks from NOESY spectra reveal double-stranded regions. Chemical shifts of protons in GH1, UH3, and UH5 of GU pairs, UH3, UH5, and AH2 of AU pairs, and GH1 of GC pairs were analyzed to identify constraints for the 5' to 3' directionality of base pairs in helices. The 5' to 3' directionality constraints were incorporated into an NMR-assisted prediction of secondary structure (NAPSS-CS) program. When it was tested on 18 structures, including nine pseudoknots, the sensitivity and positive predictive value were improved relative to those of three unrestrained programs. The prediction accuracy for the pseudoknots improved the most. The program also facilitates assignment of chemical shifts to individual nucleotides, a necessary step for determining three-dimensional structure. [ABSTRACT FROM AUTHOR]

There are large differences between the cellular environment and the conditions widely used to study RNA in vitro. SHAPE RNA structure probing in Escherichia coli cells has shown that the cellular environment stabilizes both long-range and local tertiary interactions in the adenine riboswitch aptamer domain. Synthetic crowding agents are widely used to understand the forces that stabilize RNA structure and in efforts to recapitulate the cellular environment under simplified experimental conditions. Here, we studied the structure and ligand binding ability of the adenine riboswitch in the presence of the macromolecular crowding agent, polyethylene glycol (PEG). Ethylene glycol and low-molecular mass PEGs destabilized RNA structure and caused the riboswitch to sample secondary structures different from those observed in simple buffered solutions or in cells. In the presence of larger PEGs, longer-range loop–loop interactions were more similar to those in cells than in buffer alone, consistent with prior work showing that larger PEGs stabilize compact RNA states. Ligand affinity was weakened by low-molecular mass PEGs but increased with high-molecular mass PEGs, indicating that PEG cosolvents exert complex chemical and steric effects on RNA structure. Regardless of polymer size, however, nucleotide-resolution structural characteristics observed in cells were not recapitulated in PEG solutions. Our results reveal that the cellular environment is difficult to recapitulate in vitro; mimicking the cellular state will likely require a combination of crowding agents and other chemical species. [ABSTRACT FROM AUTHOR]

Predicting the secondary structure of RNA is an intermediate in predicting RNA three-dimensional structure. Commonly, determining RNA secondary structure from sequence uses free energy minimization and nearest neighbor parameters. Current algorithms utilize a sequence-independent model to predict free energy contributions of dinucleotide bulges. To determine if a sequence-dependent model would be more accurate, short RNA duplexes containing dinucleotide bulges with different sequences and nearest neighbor combinations were optically melted to derive thermodynamic parameters. These data suggested energy contributions of dinucleotide bulges were sequencedependent, and a sequence-dependent model was derived. This model assigns free energy penalties based on the identity of nucleotides in the bulge (3.06 kcal/mol for two purines, 2.93 kcal/mol for two pyrimidines, 2.71 kcal/mol for S'-purinepyrimidine- 3', and 2.41 kcal/mol for 5'-pyrimidine-purine-3'). The predictive model also includes a 0.45 kcal/mol penalty for an A-U pair adjacent to the bulge and a .0.28 kcal/mol bonus for a G-U pair adjacent to the bulge. The new sequence-dependent model results in predicted values within, on average, 0.17 kcal/mol of experimental values, a significant improvement over the sequence-independent model. This model and new experimental values can be incorporated into algorithms that predict RNA stability and secondary structure from sequence. [ABSTRACT FROM AUTHOR]

Tau fibrils are the main proteinacious components of neurofibrillary lesions in Alzheimer disease. Although RNA molecules are sequestered into these lesions, their relationship to Tau fibrils is only poorly understood. Such understanding, however, is important, as short fibrils can transfer between neurons and nonproteinacious factors including RNA could play a defining role in modulating the latter process. Here, we used sedimentation assays combined with electron paramagnetic resonance (EPR), fluorescence, and absorbance spectroscopy to determine the effects of RNA on Tau fibril structure and growth. We observe that, in the presence of RNA, three-repeat (3R) and four-repeat (4R) Tau form fibrils with parallel, in-register arrangement of β-strands and exhibit an asymmetric seeding barrier in which 4R Tau grows onto 3R Tau seeds but not vice versa. These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents. Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin. These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain. [ABSTRACT FROM AUTHOR]

We introduce a melded chemical and computational approach for probing and modeling higher-order intramolecular tertiary interactions in RNA 2'-Hydroxyl molecular interference (HMX) identifies nucleotides in highly packed regions of an RNA by exploiting the ability of bulky adducts at the 2'-hydroxyl position to disrupt overall RNA structure. HMX was found to be exceptionally selective for quantitative detection of higher-order and tertiary interactions. When incorporated as experimental constraints in discrete molecular dynamics simulations, HMX information yielded accurate three-dimensional models, emphasizing the power of molecular interference to guide RNA tertiary structure analysis and fold refinement. In the case of a large, multidomain RNA, the Tetrahymena group I intron, HMX identified multiple distinct sets of tertiary structure interaction groups in a single, concise experiment. [ABSTRACT FROM AUTHOR]

The VS ribozyme is a catalytic RNA found within some natural isolates of Neurospora that is being used as a model system to improve our understanding of RNA structure, catalysis, and engineering. The catalytic domain contains five helical domains (SLII-SLVI) that are organized by two three-way junctions. The III-IV-V junction is required for high-affinity binding of the substrate domain (SLI) through formation of a kissing loop interaction with SLV. Here, we determine the high-resolution nuclear magnetic resonance (NMR) structure of a 47-nucleotide RNA containing the HI-IV-V junction (J345). The J345 RNA adopts a Y-shaped fold typical of the family C three-way junctions, with coaxial stacking between stems III and IV and an acute angle between stems III and V. The NMR structure reveals that the core of the III-IV -V junction contains four stacked base triples, a U-turn motif, a cross-strand stacking interaction, an A-minor interaction, and a ribose zipper. In addition, the NMR structure shows that the cCUUGg tetraloop used to stabilize stem IV adopts a novel RNA tetraloop fold, different from the known gCUUGc tetraloop structure. Using Mn2+-induced paramagnetic relaxation enhancement, we identify six Mg2+-binding sites within J345, including one associated with the cCUUGg tetraloop and two with the junction core. The NMR structure of J345 likely represents the conformation of the III-IV -V junction in the context of the active VS ribozyme and suggests that this junction functions as a dynamic hinge that contributes to substrate recognition and catalysis. Moreover, this study highlights a new role for family C three-way junctions in long-range tertiary interactions. [ABSTRACT FROM AUTHOR]

The length of the CAG-repeat region in the huntingtin mRNA is predictive of Huntington's disease. Structural studies of CAG-repeat-containing RNAs suggest that these sequences form simple hairpin structures; however, in the contest of the full-length huntingtin mRNA, CAG repeats may form complex structures that could be targeted for therapeutic intervention. We examined the structures of transcripts spanning the first exon of the huntingtin mRNA with both healthy and disease-prone repeat lengths. In transcripts with 17-70 repeats, the CAG sequences base paired extensively with nucleotides in the 5' UTR and with conserved downstream sequences including a CCG-repeat region. In huntingtin transcripts with healthy numbers of repeats, the previously observed CAG hairpin was either absent or short In contrast, in transcripts with disease-associated numbers of repeats, a CAG hairpin was present and extended from a three-helix junction. Our findings demonstrate the profound importance of sequence context in RNA folding and identify specific structural differences between healthy and disease-inducing huntingtin alleles txiat may be targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

The standard sodium concentration for RNA optical melting experiments is 1.021 M. Algorithms that predict Tm ΔG°37, and secondary structure from sequence generally rely on parameters derived from optical melting experiments performed in 1.021 M sodium. Physiological monovalent cation concentrations are much lower than 1.021 M. In fact, many molecular biology techniques require buffers containing monovalent cation concentrations other than 1.021 M. Predictions based on the 1.021 M Na+ parameters may not be accurate when the monovalent cation concentration is not 1.021 M. Here, we report thermodynamic data from optical melting experiments for a set of 18 RNA duplexes, each melted over a wide range of sodium ion concentrations (71, 121, 221, and 621 mM). Using these data and previously published data for the same sequences melted in 1.021 M. Na+, we report Tm and ΔG°37 correction factors to scale the standard 1.021 M Na+ RNA parameters to other sodium ion concentrations. The recommended Tm correction factor predicts the melting temperature within 0.7 °C, and the recommended ΔG°37 correction factor predicts the free energy within 0.14 kcal/mol. These correction factors can be incorporated into prediction algorithms that predict RNA secondary structure from sequence and provide Tm and ΔG°37 values for RNA duplexes. [ABSTRACT FROM AUTHOR]

Telomeric repeat-containing RNAs (TERRA) are transcription products of the telomeres. Human TERRA sequences containing UUAGGG repeats can form parallel-stranded G-quadruplexes. The stacking interaction of such structures was shown to be important for ligand targeting and higher-order arrangement of G-quadruplexes in long TERRA sequences. Here we report on the first high-resolution structure of a stacked G-quadruplex formed by the 10-nucleotide human TERRA sequence r(GGGUUAGGGU) in potassium solution. This structure comprises two dimeric three-layer parallel-stranded G-quadruplex blocks, which stack on each other at their 5'-ends. The adenine in each UUA loop is nearly coplanar with the 5'-end G-tetrad forming an A·(G·G·G·G)·A hexad, thereby increasing the stacking contacts between the two blocks. Interestingly, this stacking and loop conformation is different from all structures previously reported for the free human TERRA but resembles the structure previously determined for a complex between a human TERRA sequence and an acridine ligand. This stacking conformation is a potential target for drugs that recognize or induce the stacking interface. [ABSTRACT FROM AUTHOR]

The ability to predict RNA secondary structure is fundamental for understanding and manipulating RNA function. The information obtained from selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments greatly improves the accuracy of RNA secondary structure prediction. Recently, Das and colleagues [Kladwang, W., et al. (2011) Biochemistry50, 8049-8056] proposed a "bootstrapping" approach for estimating the variance and helix-by-helix confidence levels of predicted secondary structures based on resampling (randomizing and summing) the measured SHAPE data. We show that the specific resampling approach described by Kladwang et al. introduces systematic errors and underestimates confidence in secondary structure prediction using SHAPE data. Instead, a leave-data-out jackknife approach better estimates the influence of a given experimental data set on SHAPE-directed secondary structure modeling. Even when 35% of the data were left out in the jackknife approach, the confidence levels of SHAPE-directed secondary structure prediction were significantly higher than those calculated by Das and colleagues using bootstrapping. Helix confidence levels were thus underestimated in the recent study, and the resampling approach implemented by Kladwang et al. is not an appropriate metric for evaluating SHAPE-directed secondary structure modeling. [ABSTRACT FROM AUTHOR]

DEAD-box proteins are believed to participate in the folding of RNA by destabilizing RNA secondary or tertiary structures. Although these proteins bind and hydrolyze ATP, the mechanism by which nucleotide hydrolysis is coupled to helix destabilization may vary among different DEAD-box proteins. To investigate their abilities to disrupt helices and couple ATP hydrolysis to unwinding, we assayed the Saccharomyces cerevisiae ribosomal DEAD-box proteins, Dbp3p, Dbp4p, Rok1p, and Rrp3p utilizing a series of RNA substrates containing a short duplex and either a 5' or 3' single-stranded region. All four proteins unwound a 10 bp helix in vitro in the presence of ATP; however, significant dissociation of longer helices was not observed. While Dbp3p did not require a single-stranded extension to disrupt a helix, the unwinding activities of Dbp4p, Rok1p, and Rrp3p were substantially stimulated by either a 5' or 3' single-stranded extension. Interestingly, these proteins showed a clear length dependency with 3' extensions that was not observed with 5' extensions, suggesting that they bind substrates with a preferred orientation. In the presence of AMPPNP or ADP, all four proteins displayed displacement activity suggesting that nucleotide binding is sufficient to facilitate duplex disruption. Further enhancement of the strand displacement rate in the presence of ATP was observed for only Dbp3p and Rrp3p. [ABSTRACT FROM AUTHOR]

In this study, we employed a combination of steady-state and time-resolved fluorescence spectroscopy and studied the site-specific dynamics in a GTP aptamer using 2-aminopurine as a fluorescent probe. We compared the dynamics of the GTP-bound aptamer with that of the free aptamer as well as when it is denatured. GTP binding leads to an overall compaction of structure in the aptamer. The general pattern of fluorescence lifetimes and correlation times scanned across several locations in the aptamer does not seem to change following GTP binding. However, a remarkable narrowing of the lifetime distribution of the aptamer ensues following its compaction by GTP binding. Interestingly, such a "conformational narrowing" is evident from the lifetime readouts of the nucleotide belonging to the stem as well as the "bulge" part of the aptamer, independent of whether it is directly interacting with GTP. Taken together, these results underscore the importance of an overall intrinsic structure associated with the free aptamer that is further modulated following GTP binding. This work provides strong support for the "conformational selection" hypothesis of ligand binding. [ABSTRACT FROM AUTHOR]

To investigate the mechanism by which urea destabilizes RNA structure, urea-induced unfolding of four different RNA secondary and tertiary structures was quantified in terms of an m-value, the rate at which the free energy of unfolding changes with urea molality. From literature data and our osmometric study of a backbone analogue, we derived average interaction potentials (per square angstrom of solvent accessible surface) between urea and three kinds of RNA surfaces: phosphate, ribose, and base. Estimates of the increases in solvent accessible surface areas upon RNA denaturation were based on a simple model of unfolded RNA as a combination of helical and single-strand segments. These estimates, combined with the three interaction potentials and a term to account for interactions of urea with released ions, yield calculated m-values that are in good agreement with experimental values (200 mm monovalent salt). Agreement was obtained only if single-stranded RNAs were modeled in a highly stacked, A-form conformation. The primary driving force for urea-induced denaturation is the strong interaction of urea with the large surface areas of bases that become exposed upon denaturation of either RNA secondary or tertiary structure, though interactions of urea with backbone and released ions may account for up to a third of the m-value. Urea m-values for all four RNAs are salt-dependent, which we attribute to an increased extension (or decreased charge density) of unfolded RNAs with an increased urea concentration. The sensitivity of the urea m-value to base surface exposure makes it a potentially useful probe of the conformations of RNA unfolded states. [ABSTRACT FROM AUTHOR]

A misfolded form of the prion protein (PrP) is the primary culprit in mammalian prion diseases. It has been shown that nucleic acids catalyze the misfolding of cellular PrP into a scrapie-like conformer. It has also been observed that the interaction of PrP with nucleic acids is nonspecific and that the complex can be toxic to cultured cells. No direct correlation has yet been drawn between changes in PrP structure and toxicity due to nucleic acid binding. Here we asked whether different aggregation, stability, and toxicity effects are detected when nonrelated DNA sequences interact with recombinant PrP. Using spectroscopic techniques to analyze PrP tertiary and secondary structure and cellular assays to assess toxicity, we found that rPrP-DNA interactions lead to different aggregated species, depending on the sequence and size of the oligonucleotide tested. A 21-mer DNA sequence (D67) induced higher levels of aggregation and also dissimilar structural changes in rPrP, compared to binding to oligonucleotides with the same length and different nucleotide sequences or different GC contents. The rPrP-D67 complex induced significant cell dysfunction, which appears to be correlated with the biophysical properties of the complex. Although sequence specificity is not apparent for PrP-nucleic acid interactions, we believe that particular nucleic acid patterns, possibly related to GC content, oligonucleotide length, and structure, govern PrP recognition. Understanding the structural and cellular effects observed for PrP-nucleic acid complexes may shed light on the still mysterious pathology of the prion protein. [ABSTRACT FROM AUTHOR]

To better elucidate RNA structure–function relationships and to improve the design of pharmaceutical agents that target specific RNA motifs, an understanding of RNA primary, secondary, and tertiary structure is necessary. The prediction of RNA secondary structure from sequence is an intermediate step in predicting RNA three-dimensional structure. RNA secondary structure is typically predicted using a nearest neighbor model based on free energy parameters. The current free energy parameters for 2 X 3 nucleotide loops are based on a 23-member data set of 2 X 3 loops and internal loops of other sizes. A database of representative RNA secondary structures was searched to identify 2 x 3 nucleotide loops that occur in nature. Seventeen of the most frequent 2 X 3 nudeotide loops in this database were studied by optical melting experiments. Fifteen of these loops melted in a two-state manner, and the associated experimental ΔG°37,2x3 values are, on average, 0.6 and 0.7 kcal/mol different from the values predicted for these internal loops using the predictive models proposed by Lu, Turner, and Mathews [Lu, Z. J., Turner, D. H., and Mathews, D. H. (2006) Nucleic Acids Rct. 34, 4912-4924] and Chen and Turner [Chen, C., and Turner, D. H. (2006) Biochemistiy 45, 4025- 4043], respectively. These new ΔG°37253 values can be used to update the current algorithms that predict secondary structure from sequence. To improve free energy calculations for duplexes containing 2 x 3 nucleotide loops that still do not have experimentally determined free energy contributions, an updated predictive model was derived. This new model resulted from a linear regression analysis of the data reported here combined with 31 previously studied 2 X 3 nudeotide internal loops. Most of the values for the parameters in this new predictive model are within experimental error of those of the previous models, suggesting that approximations and assumptions associated with the derivation of the previous nearest neighbor parameters were valid. The updated predictive model predicts free energies of 2 X 3 nudeotide internal loops within 0.4 kcal/mol, on average, of the experimental free energy values. Both the experimental values and the updated predictive model can be used to improve secondary structure prediction from sequence. [ABSTRACT FROM AUTHOR]

Thermodynamic parameters for GU pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for 29 RNA duplexes with GU pairs to improve nearest neighbor parameters for predicting stabilities of helixes. The updated model eliminates a prior penalty assumed for terminal GU pairs. Six additional duplexes with the S'GG/3'UU motif were added to the single representation in the previous database. This revises the ΔG°37 for the S'GG/3'UU motif from an unfavorable 0.5 kcal/mol to a favorable -02 kcal/mol. Similarly, the ΔG°37 for the 5'UC/3'GU motif changes from 0.3 to -0.6 kcal/mol. The correlation coefficients between predicted and experimental ΔG°37, ΔH°, and ΔS° for the expanded database are 0.95, 0.89, and 0.87, respectively. The results should improve predictions of RNA secondary structure. [ABSTRACT FROM AUTHOR]

Precursor-mRNA splicing is catalyzed by an extraordinarily large and highly dynamic macromolecular assemblage termed the spliceosome. Detailed biochemical and structural study of the spliceosome presents a formidable challenge, but there has recently been significant progress made on this front highlighted by the crystal structure of a 10-subunit human U1 snRNP. This review provides an overview of our current understanding of the architecture of the spliceosome and the RNA–protein complexes integral to its function, the U snRNPs. [ABSTRACT FROM AUTHOR]

Currently, several models for predicting the secondary structure of RNA exist, one of which is free energy minimization using the Nearest Neighbor Model. This model predicts the lowest-free energy secondary structure from a primary sequence by summing the free energy contributions of the Watson-Crick nearest neighbor base pair combinations and any noncanonical secondary structure motif. The Nearest Neighbor Model also assumes that the free energy of the secondary structure motif is dependent solely on the identities of the nucleotides within the motif and the motif's nearest neighbors. To test the current assumption of the Nearest Neighbor Model that the non-nearest neighbors do not affect the stability of the motif, we optically melted different stem-loop oligonucleotides to experimentally determine their thermodynamic parameters. In each of these oligonucleotides, the hairpin loop sequence and the adjacent base pairs were held constant, while the first or second non-nearest neighbors were varied. The experimental results show that the thermodynamic contributions of the hairpin loop were dependent upon the identity of the first non-nearest neighbor, while the second non-nearest neighbor had a less obvious effect. These results were then used to create an updated model for predicting the thermodynamic contributions of a hairpin loop to the overall stability of the stem-loop structure. [ABSTRACT FROM AUTHOR]

The RNA duplex, (5'GACGAGUGUCA)2, has two conformations in equilibrium. The nuclear magnetic resonance solution structure reveals that the major conformation of the loop, 5'GAGU/3'UGAG, is novel and contains two unusual WatsonÂCrick/Hoogsteen GG pairs with G residues in the syn conformation, two A residues stacked on each other in the center of the helix with inverted sugars, and two bulged-out U residues. The structure provides a benchmark for testing approaches for predicting local RNA structure and a sequence that allows the design of a unique arrangement of functional groups and/or a conformational switch into nucleic acids. [ABSTRACT FROM AUTHOR]

For decades, dimethyl sulfate (DMS) mapping has informed manual modeling of RNA structure in vitro and in vivo. Here, we incorporate DMS data into automated secondary structure inference using an energy minimization framework developed for 2'-OH acylation (SHAPE) mapping. On six noncoding RNAs with crystallographic models, DMS-guided modeling achieves overall false negative and false discovery rates of 9.5% and 11.6%, respectively, comparable to or better than those of SHAPE-guided modeling, and bootatrapping provides straightforward confidence estimates. Integrating DM5-SHAPE data and including 1-cydohexyl(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT) reactivities provide small additional improvements. These results establish DMS mapping, an already routine technique, as a quantitative tool for unbiased RNA secondary structure modeling. [ABSTRACT FROM AUTHOR]

Locked nudeic acid (LNA) analogues with 24'-bridged sugars show promise in antisense applications. S-5'-Me-LNA has high RNA affinity, and modified oligonucleotides show weakened immune stimulation in vivo. Conversely, an R-5'-methyl group dramatically lowers RNA a~nity. To test the effects of S-and R-6'-methyl groups on 3'-fiuoro hexitol nucleic acid (FHNA) stability, we synthesized S-and R-6-Me-FHNA thymidine and incorporated them into oligo-2'-deoxynucleotides. As with LNA, 5-6-Me is stabilizing whereas R-6'-Me is destabiliz-ing. Crystal structures of 6'-Me-FHNA-modified DNAS explain the divergent consequences for stability and suggest convergent origins of these effects by S-and R-6-Me (FHNA) [-5-Me (LNA and RNA)] substituents. [ABSTRACT FROM AUTHOR]